The invention relates to novel core 2 xcex2-1,6-N-acetylglycosaminyltransferase nucleic acid molecules, polypeptides encoded by such nucleic acid molecules, and uses of the nucleic acid molecules and polypeptides.
The enzyme UDP-GlcNAc:Gal[xcex2] 1,3GalNAc-R (GlcNAc to GalNAc) [xcex2] 1,6-N-acetylglucosaminyltransferase (i.e. core 2 xcex2-1,6-N-acetylglycosaminyltransferase) converts core 1 (i.e. Gal[xcex2]1,3GalNAc[xcex1]-O) to core 2 structures (i.e. Gal[xcex2]1,3[GlcNAc[xcex2]1,6]GalNAc[xcex1]-O in the O-linked glycan biosynthesis pathway (Williams and Schachter, 1980 J. Biol. Chem 255:11247, 1980 and Schachter H. and Brockhausen, I, In: Allen,, H. J. and Kisailus, E. C. (eds) Glycoconjugates. Composition, Structure, and Function. Marcel Dekker, New York, pp 263-332). Core 2 GlcNAc-T activity is important in the extension of O-linked sugars with poly(N-acetyllactosamine) (i.e. repeating Gal [xcex2] 1-4GlcNAc [xcex2] 1-3). These structures have been associated with malignant transformation (Yousefi et al, 1991) and proliferative activation of lymphocytes (Higgins et al, 1991), they affect cellular adhesion (Zhu and Laine, 1985; Laferte and Dennis, 1988), and they may act as ligands for mammalian lectins (Merkle and Cummings, 1988)
Synthesis of branched, complex core 2-based O-linked structures has been found to be controlled by the relative levels of core 2 GlcNAc-T and [xcex1]-2,3 sialyl-T (Whitehouse et al, 1997) which compete for the same core 1 acceptor substrate. Therefore, core 2 is a key enzyme in the modulation of cellxe2x80x94cell interactions through glycosylation of target molecules. For example, glycosylation of PSGL-1 modulated by core 2 GlcNAc-T has been found to be a critical step for binding to P-selectin (Kumar et al, 1996; Li et al, 1996).
Expression of Core 2 GlcNAc-T in diabetic heart has also been associated with a stress-response and myocardial hypertrophy (Nishio Y. et al, J. Clin Invest 1995 October; 96(4): 1759-67). Diabetes and hyperglycemia induces core 2 GlcNAc-T gene expression specifically in cardiac myocytes of rats.
GalNAcxcex1R prevents core 2 synthesis by blocking one enzyme earlier in the O-linked pathway, and it reduces invasion and metastasis. A somatic mutation that prevents UDP-Gal transport into the Golgi blocking O- and N-linked extensions including core 2 structures causes a more severe attenuation of metastasis than a block in either pathway alone, suggesting both O-linked core 2 and N-linked branched oligosaccharides contribute to the malignant phenotype. Most recently, it was demonstrated that an increased expression of core 2 GlcNAc-T in colorectal cancer cells is closely correlated with the progression of the disease (Shimodaira K., at al 97, Cancer Res.).
The identification of new core 2 GlcNAc-transferases and nucleic acids encoding the enzymes satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of disorders mediated by the enzymes including cancer and inflammatory disorders.
The citation of any reference herein is not an admission that such reference is available as prior art to the instant invention.
The present inventors have identified novel core 2 xcex2-1,6-N-acetylglycosaminyltransferase nucleic acid molecules, and polypeptides encoded by such nucleic acid molecules. The nucleic acid molecules are herein designated xe2x80x9ccore2c GlcNAc-Txe2x80x9d or xe2x80x9ccore2c GlcNAc-Txe2x80x9d, and the polypeptides are herein designated xe2x80x9cCore 2cxe2x80x9d, xe2x80x9cCore 2c GlcNAc-Txe2x80x9d, or xe2x80x9cCore 2c GlcNAc-T Polypeptidexe2x80x9d. The core 2c GlcNAc-T nucleic acid molecules were found to be primarily expressed in the placenta, kidney, liver, and thymus.
Broadly stated the present invention contemplates an isolated Core 2c GlcNAc-T nucleic acid molecule encoding a polypeptide of the invention, including mRNAs, DNAs, cDNAs, genomic DNAs, PNAs, as well as antisense analogs and biologically, diagnostically, prophylactically, clinically or therapeutically useful variants or fragments thereof, and compositions comprising same.
The invention also contemplates an isolated Core 2c GlcNAc-T polypeptide encoded by a nucleic acid molecule of the invention a truncation, an analog, an allelic or species variation thereof, or a homolog of a polypeptide of the invention or a truncation thereof. (Truncations, analogs, allelic or species variations, and homologs are collectively referred to herein as xe2x80x9cCore 2c GlcNAc-T Related Polypeptidesxe2x80x9d). The polypeptide comprises cytosolic, transmembrane, and catalytic regions.
The nucleic acid molecules of the invention permit identification of untranslated nucleic acid sequences or regulatory sequences that specifically promote expression of genes operatively linked to the promoter regions. Identification and use of such promoter sequences are particularly desirable in instances, such as gene transfer or gene therapy, which may specifically require heterologous gene expression in a limited environment. The invention therefore contemplates a nucleic acid molecule comprising a non-coding sequence such as a 5xe2x80x2 and/or 3xe2x80x3 sequence.
The nucleic acid molecules which encode for the mature core 2c GlcNAc-T polypeptide (may include only the coding sequence for the mature polypeptide (SEQ ID NO. 1, 7 or 10); the coding sequence for the mature polypeptide and additional coding sequences (e.g. leader or secretory sequences, proprotein sequences); the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5xe2x80x2 and/or 3xe2x80x2 of the coding sequence of the mature polypeptide (e.g. SEQ ID NO. 9).
Therefore, the term xe2x80x9cnucleic acid molecule encoding a polypeptidexe2x80x9d encompasses a nucleic acid molecule which includes only coding sequence for the polypeptide as well as a nucleic acid molecule which includes additional coding and/or non-coding sequences.
The nucleic acid molecules of the invention may be inserted into an appropriate vector, and the vector may contain the necessary elements for the transcription and translation of an inserted coding sequence. Accordingly, vectors may be constructed which comprise a nucleic acid molecule of the invention, and where appropriate one or more transcription and translation elements linked to the nucleic acid molecule.
Vectors are contemplated within the scope of the invention which comprise regulatory sequences of the invention, as well as chimeric gene constructs wherein a regulatory sequence of the invention is operably linked to a heterologous nucleic acid, and a transcription termination signal.
A vector can be used to transform host cells to express a Core 2c GlcNAc-T Polypeptide or Core 2c GlcNAc-T Related Polypeptide, or a heterologous polypeptide (i.e. a polypeptide not naturally expressed in the host cell). Therefore, the invention further provides host cells containing a vector of the invention. The invention also contemplates transgenic non-human mammals whose germ cells and somatic cells contain a vector comprising a nucleic acid molecule of the invention in particular one that encodes an analog of Core 2c GlcNAc-T, or a truncation of Core 2c GlcNAc-T.
The polypeptides of the invention may be obtained as an isolate from natural cell sources, but they are preferably produced by recombinant procedures. In one aspect the invention provides a method for preparing a Core 2c GlcNAc-T Polypeptide, or a Core 2c GlcNAc-T Related Polypeptide utilizing the purified and isolated nucleic acid molecules of the invention. In an embodiment a method for preparing a Core 2c GlcNAc-T Polypeptide, or a Core 2c GlcNAc-T Related Polypeptide is provided comprising:
(a) transferring a vector of the invention comprising a nucleic acid sequence encoding a Core 2c GlcNAc-T Polypeptide, or Core 2c GlcNAc-T Related Polypeptide, into a host cell;
(b) selecting transformed host cells from untransformed host cells;
(c) culturing a selected transformed host cell under conditions which allow expression of the Core 2c GlcNAc-T Polypeptide, or Core 2c GlcNAc-T Related Polypeptide; and
(d) isolating the Core 2c GlcNAc-T Polypeptide, or a Core 2c GlcNAc-T Related Polypeptide.
The invention further broadly contemplates a recombinant Core 2c GlcNAc-T Polypeptide, or a Core 2c GlcNAc-T Related Polypeptide obtained using a method of the invention.
A Core 2c GlcNAc-T Polypeptide, or a Core 2c GlcNAc-T Related Polypeptide of the invention may be conjugated with other molecules, such as polypeptides, to prepare fusion polypeptides or chimeric polypeptides. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion polypeptides.
The invention further contemplates antibodies having specificity against an epitope of a Core 2c GlcNAc-T Polypeptide, or a Core 2c GlcNAc-T Related Polypeptide of the invention. Antibodies may be labeled with a detectable substance and used to detect polypeptides of the invention in biological samples, tissues, and cells.
The invention also permits the construction of nucleotide probes that are unique to nucleic acid molecules of the invention and/or to polypeptides of the invention. Therefore, the invention also relates to a probe comprising a sequence encoding a polypeptide of the invention, or a portion (i.e. fragment) thereof. The probe may be labeled, for example, with a detectable substance and it may be used to select from a mixture of nucleic acid molecules a nucleic acid molecule of the invention including nucleic acid molecules coding for a polypeptide which displays one or more of the properties of a polypeptide of the invention.
In accordance with an aspect of the invention there is provided a method of, and products for (i.e. kits), diagnosing and monitoring conditions mediated by core 2c GlcNAc-transferases by determining the presence of nucleic acid molecules and polypeptides of the invention.
Still further the invention provides a method for evaluating a test compound for its ability to modulate the biological activity of a Core 2c GlcNAc-T Polypeptide, or a Core 2c GlcNAc-T Related Polypeptide of the invention. For example, a substance which inhibits or enhances the catalytic activity of a Core 2c GlcNAc-T Polypeptide, or a Core 2c GlcNAc-T Related Polypeptide may be evaluated. xe2x80x9cModulatexe2x80x9d refers to a change or an alteration in the biological activity of a polypeptide of the invention. Modulation may be an increase or a decrease in activity, a change in characteristics, or any other change in the biological, functional, or immunological properties of the polypeptide.
Compounds which modulate the biological activity of a polypeptide of the invention may also be identified using the methods of the invention by comparing the pattern and level of expression of a nucleic acid molecule or polypeptide of the invention in biological samples, tissues and cells, in the presence, and in the absence of the compounds.
Methods are also contemplated that identify compounds or substances (e.g. polypeptides) which interact with core 2c regulatory sequences (e.g. promoter sequences, enhancer sequences, negative modulator sequences, see SEQ ID NO. 9).
The nucleic acid molecules, polypeptides, and substances and compounds identified using the methods of the invention, may be used to modulate the biological activity of a Core 2c GlcNAc-T Polypeptide, or a Core 2c GlcNAc-T Related Polypeptide of the invention, and they may be used in the treatment of conditions mediated by core 2c GlcNAc-Transferases such as inflammatory disorders, liver disorders, kidney disorders, disorders of the thymus, and proliferative disorders such as cancer. Accordingly, the nucleic acid molecules, polypeptides, substances and compounds may be formulated into compositions for administration to individuals suffering from one or more of these conditions. Therefore, the present invention also relates to a composition comprising one or more of a polypeptide, nucleic acid molecule, or substance or compound identified using the methods of the invention, and a pharmaceutically acceptable carrier, excipient or diluent. A method for treating or preventing these conditions is also provided comprising administering to a patient in need thereof, a composition of the invention.
The present invention in another aspect provides means necessary for production of gene-based therapies directed at the placenta, liver, kidney, and thymus. These therapeutic agents may take the form of polynucleotides comprising all or a portion of a nucleic acid molecule of the invention comprising a regulatory sequence of core 2c GlcNAc-T placed in appropriate vectors or delivered to target cells in more direct ways.
Having provided a novel Core 2c GlcNAc-T Polypeptide, and nucleic acids encoding same, the invention accordingly further provides methods for preparing oligosaccharides e.g. two or more saccharides including sLex antigens. In specific embodiments, the invention relates to a method for preparing an oligosaccharide comprising contacting a reaction mixture comprising a sugar donor, and an acceptor in the presence of a Core 2c GlcNAc-T Polypeptide, or a Core 2c GlcNAc-T Related Polypeptide of the invention.
In accordance with a further aspect of the invention, there are provided processes for utilizing polypeptides or nucleic acid molecules, for in vitro purposes related to scientific research, synthesis of DNA, and manufacture of vectors.
These and other aspects, features, and advantages of the present invention should be apparent to those skilled in the art from the following drawings and detailed description.